Analysis of the mechanisms that underlie absorption of botulinum toxin by the inhalation route.

Botulinum toxin is a highly potent oral and inhalation poison, which means that the toxin must have an efficient mechanism for penetration of epithelial barriers. To date, three models for toxin passage across epithelial barriers have been proposed: (i) the toxin itself undergoes binding and transcytosis; (ii) an auxiliary protein, HA35, transports toxin from the apical to the basal side of epithelial cells; and (iii) an auxiliary protein, HA35, acts on the basal side of epithelial cells to disrupt tight junctions, and this permits paracellular flux of toxin. These models were evaluated by studying toxin absorption following inhalation exposure in mice. Three types of experiments were conducted. In the first, the potency of pure neurotoxin was compared with that of progenitor toxin complex, which contains HA35. The results showed that the rate and extent of toxin absorption, as well as the potency of absorbed toxin, did not depend upon, nor were they enhanced by, the presence of HA35. In the second type of experiment, the potencies of pure neurotoxin and progenitor toxin complex were compared in the absence or presence of antibodies on the apical side of epithelial cells. Antibodies directed against the neurotoxin protected against challenge, but antibodies against HA35 did not. In the final type of experiment, the potency of pure neurotoxin and toxin complex was compared in animals pretreated to deliver antibodies to the basal side of epithelial cells. Once again, antibodies directed against the neurotoxin provided resistance to challenge, but antibodies directed against HA35 did not. Taken collectively, the data indicate that the toxin by itself is capable of crossing epithelial barriers. The data do not support any hypothesis in which HA35 is essential for toxin penetration of epithelial barriers.

Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis.

Evidence that botulinum toxin receptors on epithelial cells and neuronal cells are not identical: implications for development of a non-neurotropic vaccine.

Botulinum toxin typically interacts with two types of cells to cause the disease botulism. The toxin initially interacts with epithelial cells in the gut or airway to undergo binding, transcytosis, and delivery to the general circulation. The toxin then interacts with peripheral cholinergic nerve endings to undergo binding, endocytosis, and delivery to the cytosol. The receptors for botulinum toxin on nerve cells have been identified, but receptors on epithelial cells remain unknown. The initial toxin binding site on nerve cells is a polysialoganglioside, so experiments were performed to determine whether polysialogangliosides are also receptors on epithelial cells. A series of single mutant and dimutant forms of the botulinum toxin type A binding domain (HC50) were cloned and expressed. One of these (dimutant HC50 A(W1266L,Y1267S)) was shown to have lost its ability to bind nerve cells (phrenic nerve-hemidiaphragm preparation), yet it retained its ability to bind and cross human epithelial monolayers (T-84 cells). In addition, the wild-type HC50 and the dimutant HC50 displayed the same ability to undergo binding and transcytosis (absorption) in a mouse model. The fact that the dimutant retained the ability to cross epithelial barriers but did not possess the ability to bind to nerve cells was exploited to create a mucosal vaccine that was non-neurotropic. The wild-type HC50 and non-neurotropic HC50 proved to be comparable in their abilities to: 1) evoke a circulating IgA and IgG response and 2) evoke protection against a substantial challenge dose of botulinum toxin.

Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

BACKGROUND: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

The role of systemic handling in the pathophysiologic actions of botulinum toxin.

The ability of botulinum toxin to poison cholinergic nerve transmission is a dynamic phenomenon that involves not only the actions of the toxin on the body but also the actions of the body on the toxin. The former has been the subject of intense research, whereas the latter has received almost no attention. Therefore, a series of studies were performed to characterize systemic handling of botulinum toxin. The results indicated that the toxin reaches the general circulation (transcytosis across epithelial cells) without obvious changes in structure or biological activity. The general circulation acts as a holding compartment until there is adequate fractional distribution to neuromuscular junctions to produce blockade of transmission. During its transit through this compartment, the toxin 1) undergoes little biotransformation, 2) does not accumulate significantly in circulating cells, and 3) remains largely in the free state. In naive animals, the t(1/2) for toxin in the general circulation is approximately 10 h, and at any given point in time, there is little uptake in nontarget organs (liver, kidney, heart, and lung). In immunized animals, toxin clearance from the general circulation is rapid, and there is substantial accumulation of antibody-antigen complexes in liver. Thus, enhanced clearance from the circulation is a major mechanism by which active immunization can protect against poisoning.

Trivalent vaccine against botulinum toxin serotypes A, B, and E that can be administered by the mucosal route.

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.

An initial assessment of the systemic pharmacokinetics of botulinum toxin.

Botulinum toxin is an extraordinarily potent molecule that has an unusually long duration of action. Despite this, there is little information available on natural mechanisms for metabolism or elimination and virtually no information on pharmacologically induced mechanisms for metabolism and elimination. Therefore, a number of experiments were performed on laboratory animals that addressed two major issues: 1) the effect of blood on the structure, function, and biologic half-life of the toxin, and 2) the effect of neutralizing antibodies on half-life and elimination of circulating toxin. In the first series of studies, the metabolic transformation of toxin was assessed by incubating it in blood for varying lengths of time. At each time point, aliquots were examined to determine the amount of toxin, the structure of toxin, the catalytic activity of toxin, and the neuromuscular blocking activity of toxin. This work demonstrated that blood did not alter any characteristic of the toxin molecule. Experiments were also done in which toxin was administered to mice and rats at doses that produced clinical poisoning. The results demonstrated that the elimination half-life for native (nonmetabolized) toxin in blood and serum was 230 to 260 min. During the second series of studies, the rate of elimination of circulating toxin was studied in the presence of antibodies directed against the carboxyl-terminal half of the toxin molecule. This work demonstrated that neutralizing antibodies 1) enhanced clearance of toxin from the circulation and 2) enhanced tissue accumulation of toxin, particularly in liver and spleen.